Evaluating and forecasting the associated main flavor components in Baijiu (Chinese distilled spirits) with alcohol metabolism and hangover symptoms through mice acute withdrawal model

Abstract Over the past few decades, more alcohol‐problem concerns focused on reducing the risk of hangover caused by the alcoholic beverages over‐consumption. Chinese distilled spirits (Baijiu) is one of the most favorite alcoholic beverages. The intention of this study is to explore the associations of main flavor components in Baijiu and hangover symptoms using mice acute alcohol withdrawal model. The behaviors of each mouse were assessed by open‐field tests using separate groups of mice with the treatment of sauce‐aroma Baijiu, light‐aroma Baijiu, strong‐aroma Baijiu, pure alcohol, and distilled water, respectively. The behavioral data including total move distance and immobile time were used as indicators for the evaluation of the liquor intoxicating effects. Alcohol and acetaldehyde concentrations in mice plasma and the neurotransmitter contents of GABA and Glu in mice cerebellum were detected afterward. The results showed that the mice with the treatment of Baijiu samples displayed unusual exciting behaviors including increased alcohol metabolization with alleviating drunken and hangover symptoms, compared with that of pure alcohol control groups after 2–4 h. Moreover, the sauce‐aroma Baijiu treatment group showed lessening intoxicated symptoms than those of light‐aroma Baijiu and strong‐aroma Baijiu. In addition, there were significant differences between Baijiu and pure alcohol treatment groups at the inhibitory neurotransmitter GABAergic levels and its receptor GABA‐AR1 activating levels in the mice neuron cells. Furthermore, the Principal Component Analysis (PCA) analysis inferred that the flavor compounds acetic acid, ethyl acetate, ethyl lactate, and 1‐propanol in the sauce‐aroma Baijiu were played the major roles in the drunk behaviors that caused by the hangover. While, the acetic acid in the sauce‐aroma Baijiu was speculated as a major flavor component to accelerate the alcohol metabolism and retard hangover symptoms.


| INTRODUC TI ON
Baijiu (Chinese distilled spirits or Chinese liquor) is normally fermented and distilled from sorghum alone or a mixture of corn, rice, wheat, peas, millet, and sorghum, which is famous for its brewing history (Liu & Sun., 2018). The three important typical flavor Baijiu types are sauce-aroma (Jiangxiangxing), strong-aroma (Nongxiangxing), and light-aroma (Qingxiangxing), which are more widely distributed in Guizhou province, Sichuan, Jiangsu and Anhui province, Shanxi and Hebei province, respectively (Wei et al., 2020). Baijiu is reported to contain more than 2047 kinds of flavor compounds, which are produced from the mixed fermentation and distillation of microbial starters "jiuqu" and grains (Sun et al., 2021). Alcohols, esters, aldehydes, and acids are the four abundant kinds of flavors in Baijiu. The category and content of these substances vary greatly in different Baijiu aroma types (Cai et al., 2019). Primarily, in strong-aroma Baijiu, the concentration of ethyl hexanoate and hexanoic acid is higher than that of other aroma compounds (Fan & Qian, 2006). In lightaroma Baijiu, ethyl acetate is the main flavor substance followed by ethyl lactate (Gao et al., 2014;Niu et al., 2017). The flavor compounds are more diverse and complicated in sauce-aroma Baijiu. The organic acids content, such as the acetic and lactic acid, display the highest levels in the than other Baijiu aroma types (Fan et al., 2011).
The alcohol hangover has substantial economic and health consequences. The obvious symptoms are dullness, weakness, incoordination, fatigue, sleepiness, thirst, headache, and mood change (Penning et al., 2010;. Asian may experience more severe hangover symptoms, correlating to some extent with the presence of the aldehyde dehydrogenase (ALDH2) gene genetic variants (Wall et al., 2000). The alcohol and its metabolites acetaldehyde, immune, hormonal, and dehydration-related factors of physiological changes and cognitive functions are the determinants of hangover severity (Mackus et al., 2020). The congeners such as methanol, l-propanol, 1-butanol, 2-butanol, iso-butanol, 2-methyl-butanol, 3-methyl-butanol in alcoholic beverages were suggested to affect hangover severity . However, alcohols without congeners were also found to produce hangovers .
In China, nearly 20% of drinkers are described to not experience hangover symptoms despite heavy drinking of Baijiu. They attributed the reason for the relating to the different flavor compounds changes in production processing of Chinese and non-Chinese liquors, such as fermentation, distillation, aging, blending processes, raw materials, environmental conditions, brewing microorganisms, and fermentation starters. Furthermore, the balance and coordination of these volatile compounds not only affect the sensory quality but also affect the symptomatic relief of hangover. Nevertheless, current data comparing alcoholic beverages are limited, and little evidence indicates any effective components to prevent the hangover symptoms in Baijiu.
The similarity in biology and symptoms between the animal model and the human hangovers is generally acknowledged as previous studies described (Lucas et al., 2018). In this work, the mice acute withdrawal model was established to investigate the associations with main flavor compounds in strong-aroma Baijiu, light-aroma Baijiu, sauce-aroma Baijiu, and pure alcohol on alcohol metabolism and hangover symptoms.

| Baijiu samples
All the Baijiu samples were commercial liquor products that were purchased from the local markets (Beijing, China). The information for the Baijiu samples used in this study was listed in Table 1. The pure alcohol sample for the same ethanol content was prepared by the solution of commercial vodka (Spirytus rektyfikowany, Poland, 96%, ABV) with distilled water.

| Animals
The animals used in this study were C57BL/6J wide-type (WT) male mice with 65 days of age and an average body weight of 20 g, which were housed under standardized condition. The mice in the sauce-aroma Baijiu was speculated as a major flavor component to accelerate the alcohol metabolism and retard hangover symptoms.

| Open-field behavioral assessment
In this study, mice after-drinking behaviors assessment was utilized by Open-field. Mice were individually placed in a 30 cm × 70 cm × 47 cm arena with opaque walls. Sessions were video recorded and analyzed using ANY-maze video tracking software (Stoelting, Wood Dale, IL).

| Blood alcohol concentration determination
After the locomotor activity experiment, the animals were sacrificed directly. The blood (~0.5 ml) was then extracted via cardiac puncture. The blood was collected in sodium fluoride containing tubes to stop further alcohol metabolism. Then, these blood samples were centrifuged at 840 × g/rcf for 10 min, the serum was collected and aliquoted. The alcohol and its metabolites acetaldehyde concentration in the plasma were determined using headspace gas chromatography methods previously reported (Heit et al., 2016).

| Neurotransmitter contents and immunofluorescence analysis
The Baijiu, distilled water, and pure alcohol were given to the mice, respectively. Two hours later, the cerebellums in the mice brain were excised. Homogenates were generated using homogenizer and RIPA buffer. The neurotransmitter contents of GABA and Glu were measured using commercial ELISA kits (Shanghai enzyme-linked immunosorbent biotechnology co., Ltd, China). To determine the GABA extents in the neuron cells activation level, immunofluorescence analysis was conducted using surface GABABR1 staining (1/200 dilution; Abcam, ab55051). Image analysis platform was used to analyze the features.

| Baijiu flavor analysis
Volatile flavors in Baijiu including fusel alcohols, esters, and aldehydes were analyzed by the HS-SPME GC-MS methods according Compounds were identified by comparing the standard curve with those of pure standards under the same conditions. The identification and quantification were carried out by comparing the retention times and standard curves (R 2 > 0.99) with those of known pure compounds.

| Statistical analysis and multiple linear regression analysis
Data were analyzed and graphed using Prism version 8. 3

| Open-field behavioral assessment in alcoholized mice
Behavioral changes are the key measurement criteria of evaluating different aspects of alcohol hangover in rodents (Karadayian & Cutrera, 2013;Lucas et al., 2018). The open-field test has several merits in this regard as it monitors rodent behaviors in a novel environment with the ability to simultaneously measure exploration, anxiety-like behaviors, and general locomotion. These time course models can be used to definitively correlate variables of interest to hangover severity over time (Je et al., 2021). This type of model could provide valuable insight into how changing biochemical markers may influence or contribute to alcohol hangover (Bustamante et al., 2012;Juarez et al., 2017). In this study, for the majority of behavioral measures considered, the covered locomotion distance and immobile time persisted for 24 h were the major measurement of after-drinking behaviors in mice. The ratios of after-drinking motor performance to that of before-drinking were displayed in Figure 1.

| Blood alcohol and acetaldehyde concentrations assessment in alcoholized mice
The alcohol hangover refers to the combination of negative mental and physical symptoms which can be experienced after a single episode of alcohol consumption, starting when blood alcohol concentration (BAC) approaches zero . The rate of alcohol metabolism proposed to be an important determinant of hangover severity (Mackus et al., 2020). Measuring alcohol and its metabolites concentration to indicate a hangover state was a common method used in the alcohol hangover model (Penning et al., 2010;Tipple et al., 2016;Palmer et al., 2019). In this study, the blood alcohol and acetaldehyde concentrations were determined 2, 4, 8, and 24 h after C57BL6 mice were dosed intragastrically with pure alcohol, distilled water, and different Baijiu aroma types (Figure 2). The results demonstrated that at 24-h post-administration with all the alcoholic beverages, the BAC was returning to zero. Notably, the mice behavioral data displayed that the pure alcohol and Baijiu treatment groups did not return to normal at this time point, and still experienced a movement retardation than the water control. After intragastric filling for

F I G U R E 1
The ratios of the C57BL6 mice (n = 5) behavioral parameters at 2, 4, 8, and 24 h after intragastric administration to that of 0 h before intragastric administration with saucearoma Baijiu type, strong-aroma Baijiu type, light-aroma Baijiu type, pure alcohol, and distilled water, respectively. (Figure 1a, mobile distance (m ± SEM), Figure 1b, immobile time (s ± SEM))

| Main flavor compounds in Baijiu association analysis
Some compounds within alcoholic beverages are produced during distillation and fermentation, which may contribute to the severity of hangover induced by alcohol .  -propanol, up to more than 1000 mg/L in sauce-aroma Baijiu, far more than lightaroma Baijiu and strong-aroma Baijiu. It was previously reported that fusel alcohols and aldehydes in alcoholic beverages, such as F I G U R E 2 Measures of blood alcohol and acetaldehyde concentration analyzed 2, 4, 8, and 24 h after C57BL6 mice (n = 5) were dosed intragastrically with Baijiu, pure alcohol, and distilled water, respectively. (Figure 2a, blood alcohol concentration, Figure 2b, blood acetaldehyde concentration) whisky, brandy, wine, beer, and Chinese Baijiu, had performanceenhancing effects on the intoxicating degree (Hisako et al., 2003;Wu et al., 2016;Xiao et al., 2018). However, the functions of esters and organic acids in Baijiu on alcoholism have not been proved.
To find the existing relationship between negative behaviors data in experimental drunken mice post-alcohol administration at 2-h time point and the contents of the mentioned three main flavor components, the Principal Component Analysis (PCA) was used in this study. These two methods are simple algorithms widely used to explore the correlation between independent variables and the dependent variable Wu et al., 2016). was mainly related to the sauce-aroma Baijiu and had a further relationship from the immobile moving time, which was predicted to be positively affected on reducing hangover severity.

Sauce-aroma Baijiu is becoming increasingly accepted in
Chinese for the moment. Many drinkers generally reflected that there were little hangover symptoms after heavy drinking of sauce-aroma Baijiu. Several previous studies speculated positive correlation between the active no-alcoholic components such as pyrazine and their pharmacological effects in Sauce-aroma Baijiu . However, these active components were low contained extremely in Baijiu (Zhou et al., 2020). The literature F I G U R E 3 (a) Measures of neurotransmitter Glu and GABA levels in the cerebellum of mice brain analyzed 2 h after C57BL6 mice (n = 5) were dosed intragastrically with Baijiu, pure alcohol, and distilled water, respectively. (b) GABA-AR expression levels in the neuron cells of cerebellum after C57BL6 mice were dosed intragastrically with Baijiu, pure alcohol, and distilled water after 2 h, respectively. has not yet substantiated their positive impacts by experiments in the whole alcohol beverages intake currently. Baijiu is a distilled liquor, but its production combines the two distinctive processes of fermentation and distillation. These non-volatile acids in Baijiu were from the fermented grains by acidogenic bacteria (Liu & Sun, 2018). Most studies illustrated that the lower content of acids in the no-alcoholic beverages and fermented alcoholic beverages such as wine or beer have positive impacts on health (Valentine et al., 2021;Rhee et al., 2011). This study first demonstrated that the acids in sauce-aroma Baijiu were much more than both lightaroma and strong-aroma Baijiu, presuming it probably had positive effect on reducing hangover severity. The result might be inferred to instructive significance for the improvement of hangover severity from the brewing and blending process in Baijiu.

| CON CLUS IONS
Alcohol hangover is commonly considered as a public health concern in the world. General Chinese population may experience more severe symptoms compared to European and African ancestries, due to the marked lower clearance rate of alcohol. The two genetic variants in the Chinese alcohol metabolizing-related genes ALDH2 (rs671) and ADH1B (rs1229984 and rs2066702) that greatly alter alcohol metabolism and had a higher risk of acetaldehyde accumulation and esophageal cancer (Iona et al., 2019 GABA and Glu were displayed to correlate with intensity of behavior symptoms in the pure alcohol treatment group. The PCA analysis suggested that the main flavor compounds acetic acid in the saucearoma Baijiu had positive effects on preventing alcohol hangover for motor performance. Furthermore, the mice that were given the sauce-aroma Baijiu exhibited lessening intoxicated symptoms than those of light-aroma and strong-aroma Baijiu. The results suggested that the organic acids in the sauce-aroma Baijiu was of great importance to accelerate the alcohol metabolism. Previous study had revealed that the organic acid fraction in Japanese Sake, particularly L-lactic acid, gluconic acid, and pyruvic acid, played important roles in potentiating the GABAA receptor-mediated response which led to significant anxiolytic effects (Hanae et al., 2017). Notably, the role of GABA in alcohol responsiveness has been proved in animal models.
However, it is not obvious whether the organic acid fraction in the Baijiu directly mediates the response. To elucidate it, further analysis is required.

AUTH O R S' CO NTR I B UTI O N S
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACK N OWLED G M ENT
We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author upon reasonable request.